Clostridium difficile is a Gram-positive, spore forming anaerobic bacterium that colonizes the intestinal tract of humans thus leading to C. difficile infections (CDI). CDI has become the most commonly diagnosed cause of hospital-acquired diarrhea, particularly in the risk groups including elderly and immunodeficient patients as well as those receiving antibiotic treatment. A steep rise in CDI incidents over the past decade is attributed to the emergence of the hypervirulent, and now predominant strain ribotype 27, causing epidemic outbreaks with increased morbidity, mortality and high relapse rates. The costs to treat patients have greatly increased, particularly in the case of recurring CDI. Preventive methods, such as vaccination of risk groups, may be useful and cost-efficient means to avoid future infections. Although vaccination against C. difficile should be economically feasible (B. Y. Lee et al., Vaccine, 2010, 28, 5245) a vaccine has not yet been developed.
Carbohydrates exposed on the cell-surface of pathogens are often immunogenic and constitute potential candidates for vaccine development. When covalently connected to carrier proteins, carbohydrate antigen vaccines can elicit a long lasting T-cell dependent protection (C. Snapper and J. Mond, J. Immunol., 1996, 157, 2229). Several vaccines containing carbohydrates, isolated from biological sources, are in routine use (G. Ada and D. Isaacs, Clin. Microbiol. Infect., 2003, 9, 79). Vaccines based on synthetic carbohydrate antigens against bacteria, viruses, parasites and cancer are currently in preclinical and clinical development (a) R. D. Astronomo and D. R. Burton, Nature Rev., 2010, 9, 308; b) M.-L. Hecht, P. Stallforth, D. V. Silva, A. Adibekian and P. H. Seeberger, Curr. Opin. Chem. Biol., 2009, 13, 354).
The chemical structure of two C. difficile cell-surface polysaccharides, PS-I and PS-II has been elucidated recently (J. Ganeshapillai et al., Carbohydr. Res., 2008, 343, 703; WO 2009/033268 A1). Initial focus has been turned towards the PS-II hexasaccharide antigen that is believed to be common to several C. difficile strains (a) E. Danieli et al., Org. Lett., 2010, 13, 378; b) M. Oberli, M.-L. Hecht, P. Bindschädler, A. Adibekian, T. Adam and P. H. Seeberger, Chem. Biol., 2011, 18, 580). The synthetic PS-II hapten is immunogenic when conjugated to a carrier protein and antibodies found in the stool of C. difficile patients bind to the synthetic PS-II hexasaccharide (Oberli et al., ibid.). The pentasaccharide phosphate repeating unit PS-I was reported as [→4)-α-Rhap-(1→3)-β-Glcp-(1→4)-[α-Rhap-(1→3)]-α-Glcp-(1→2)-α-Glcp-(1→P] and it is suggested to be specific for the strain ribotype 27.
In conclusion, the pathogen C. difficile represents a major risk for patients and causes significant costs to health care systems. Unfortunately, however, currently no licensed vaccine against C. difficile is available.
Thus, a main object of the present invention is to provide novel and effective means to prevent and/or to treat C. difficile associated diseases, in particular related to the hypervirulent strain ribotype 027. A further object is to provide novel and effective means to detect C. difficile in a sample and/or a C. difficile infection in a subject. A further object is to provide novel and effective means to identify a certain strain of C. difficile in a sample and/or a C. difficile infected subject. A further object is to provide novel and effective standards for immunoassays for the detection of C. difficile. 
The present inventors succeeded in the first total synthesis of a pentasaccharide derived from the repeating unit of the C. difficile polysaccharide PS-I, and its conjugation to the diphtheria toxoid Crm197.
Consequently, the above main object of the invention is achieved by providing the synthetic oligosaccharide, in particular pentasaccharide, the oligosaccharide-protein conjugate and the composition according to the invention. Further objects are achieved by providing the antibody, the methods of detection and identification, and the methods of synthesis according to the invention.